- Remove slides and immediately coverslip with 3–5 drops of
EcoMount mounting medium. Be cautious to avoid trapping
air bubbles, as they may interfere with image capture. - Allow slides to dry on the bench for 5–10 min.
- Image slides on brightfield to capture signal. Figure4 shows
representative images of microRNA-101a (green) andfosab,a
target gene (red) on adult zebrafish regenerating hearts [11].
4 Notes
- Custom RNA probes are designed by Advanced Cell Diagnos-
tics with propriety RNAscope amplification technology. This
technology is based on synthesis of multiple RNA probes to
hybridize across a target sequence. Upon hybridization, the
signal is enhanced through a series of amplification steps and
detected either by fluorescence or by chromogenic enzymatic
reactions (Fig.1). In this example, I ordered a probe directed
against 500-bp of the microRNA primary sequence and 800-
bp of a target gene. - Advanced Cell Diagnostics (ACD) provides a 50concen-
trated wash buffer that needs to be diluted with water to 1
working concentration. We routinely use 1PBT as a substi-
tute and have not observed changes to signal intensity - In my experience ~200μL is sufficient to cover all sections. Mix
contents by pipetting up and down 3–5 times before use. Once
the working solution is made, it must be used within 15 min. - To create a hybridization chamber, select an appropriate size
Tupperware with a secure lid. Cut 5 mL serological pipettes to
the length of a Tupperware and attach them onto the bottom
surface in parallel at a distance that will support your slides,
with laboratory tape (Fig.2). During hybridization, it is impor-
tant that you place water soaked paper towels in the bottom of
the Tupperware to create a humidified chamber. All incuba-
tions are performed in a humidified chamber at 40C unless
otherwise noted. - It is important to not overfix the tissue as this may induce
nonspecific cross-linking, thereby interfering with RNA
detection. - The tissue will sink to the bottom of the centrifuge tube when
equilibration is complete. Residual 4% PFA does not interfere
with subsequent tissue processing. - The clear Tissue Freezing Medium will turn into a white solid
substance indicating that the tissue block has solidified. - It is critical to use Superfrost/Plus coated slides as sections
adhere more efficiently.
206 Viravuth P. Yin